Biotinidase is the enzyme that cleaves biotin from the final products of the proteolytic degradation of biotin-dependent carboxylases, thus recycling the vitamin. Biotinidase activity is deficient in most children with late-onset multiple carboxylase defeiciency. Affected individuals may exhibit neurologic and cutaneous features including seizures, hypotonia, ataxia, skin rash, alopecia and developmental delay, which may progress to come and ultimately death. All children with biotinidase deficiency who have been treated with biotin have improved clinically. Since the disorder met the major criteria for inclusion in newborn screening programs, we developed a simple test for determining biotinidase activity using the same blood-soaked filter paper samples used in most programs. We have been screening all the newborn infants born in Virginia since January 24, 1984 and have detected three newborns with the deficiency and two affected siblings of one of these infants. Based on our results more than a dozen states and ten foreign countries have started or will start similar newborn screening programs. Several of these programs have already detected newborns with the enzyme deficiency. We proposed to collaborate with designated physicians in states that are currently screening for biotinidase deficiency and physicians who have identified symptomatic individuals to obtain a deficiency and physicians who have identified symptomatic individuals to obtain a better understanding of the initial features and natural history of the disorder. Clinical and biochemical evaluations will be performed at regular intervals on infants and children with biotinidase deficiency detected by newborn screening or who have been identified after developing symptoms. Evaluation of their family members, particularly siblings, should provide insight into the variation of expression of the disorder and the possible existence of benign variants. Moreover, this work will provide information about the possible manifestations in heterozygotes for the disorder and the potential adverse effects of biotin treatment.